Raman spectroscopy is a well-known technique for analyzing molecules or materials. In conventional Raman spectroscopy, high intensity monochromatic radiation provided by a radiation source, such as a laser, is directed onto an analyte (or sample) that is to be analyzed. In Raman spectroscopy, the wavelength of the incident radiation typically is varied over a range of wavelengths within or near the visible region of the electromagnetic spectrum. A majority of the photons of the incident radiation are elastically scattered by the analyte. In other words, the scattered photons have the same energy, and thus the same wavelength, as the incident photons. However, a very small fraction of the photons are inelastically scattered by the analyte. Typically, only about 1 in 107 of the incident photons are inelastically scattered by the analyte. These inelastically scattered photons have a different wavelength than the incident photons. This inelastic scattering of photons is termed “Raman scattering”. The Raman scattered photons can have wavelengths less than, or, more typically, greater than the wavelength of the incident photons.
When an incident photon collides with the analyte, energy can be transferred from the photon to the molecules or atoms of the analyte, or from the molecules or atoms of the analyte to the photon. When energy is transferred from the incident photon to the analyte, the Raman scattered photon will have a lower energy and a corresponding longer wavelength than the incident photon. These Raman scattered photons having lower energy than the incident photons are collectively referred to in Raman spectroscopy as the “Stokes radiation.” A small fraction of the analyte molecules or atoms can be in an energetically excited state when photons are incident thereon. When energy is transferred from the analyte to the incident photon, the Raman scattered photon will have a higher energy and a corresponding shorter wavelength than the incident photon. These Raman scattered photons having higher energy than the incident photons are commonly referred to in Raman spectroscopy as the “anti-Stokes radiation.” The Stokes radiation and the anti-Stokes radiation collectively are referred to as the Raman scattered radiation or the Raman signal.
The Raman scattered radiation is detected by a detector that typically includes a wavelength-dispersive spectrometer and a photomultiplier for converting the energy of the impinging photons into an electrical signal. The characteristics of the electrical signal are at least partially a function of both the energy of the Raman scattered photons as evidenced by their wavelength, frequency, or wave number, and the number of the Raman scattered photons as evidenced by the intensity of the Raman scattered radiation. The electrical signal generated by the detector can be used to produce a spectral graph illustrating the intensity of the Raman scattered radiation as a function of the wavelength of the Raman scattered radiation. Analyte molecules and materials generate unique Raman spectral graphs. The unique Raman spectral graph obtained by performing Raman spectroscopy can be used for many purposes including identification of an unknown analyte, or determination of physical and chemical characteristics of a known analyte.
Raman scattering of photons is a weak process. As a result, powerful, costly laser sources typically are used to generate high intensity incident radiation to increase the intensity of the weak Raman scattered radiation for detection. Surface-enhanced Raman spectroscopy (SERS) is a technique that allows for enhancement of the intensity of the Raman scattered radiation relative to conventional Raman spectroscopy. In SERS, the analyte molecules typically are adsorbed onto or placed adjacent to what is often referred to as a SERS-active structure. SERS-active structures typically include a metal surface or structure. Interactions between the analyte and the metal surface may cause an increase in the intensity of the Raman scattered radiation. The mechanism by which the intensity of the Raman scattered radiation is enhanced is not precisely understood.
Several types of metallic structures have been employed in SERS techniques to enhance the intensity of Raman scattered radiation that is scattered by analyte molecules adjacent thereto. Some examples of such structures include electrodes in electrolytic cells, metal colloid solutions, and metal substrates such as a roughened metal surface or metal “islands” formed on a substrate. For example, it has been shown that adsorbing analyte molecules onto or near a specially roughened metal surface made from gold or silver can enhance the Raman scattering intensity by factors of between 103 and 106.
Raman spectroscopy recently has been performed employing randomly oriented nanoparticles, such as nanometer scale needles, particles, and wires, as opposed to a simple roughened metallic surface. The process of performing Raman spectroscopy utilizing structures comprising nanoparticles to enhance the intensity of Raman scattered radiation will be referred to herein as nano-enhanced Raman spectroscopy (NERS). The intensity of the Raman scattered photons from a molecule adsorbed on such a nanostructure can be increased by factors as high as 1016. At this level of sensitivity, NERS has been used to detect single molecules. Detecting single molecules with high sensitivity and molecular specificity is of great interest in the fields of chemistry, biology, medicine, pharmacology, and environmental science.
Hyper-Raman spectroscopy is another Raman spectroscopy technique that involves detecting the shift from the second order harmonic of the excitation frequency. The hyper-Raman scattered radiation is Raman-shifted relative to integer multiples of the frequency of the incident electromagnetic radiation. Hyper-Raman scattered radiation can provide information about the analyte that cannot be obtained from normal Raman spectroscopy. The intensity of the hyper-Raman scattered radiation, however, is even less than the intensity of the Raman scattered radiation. As a result, hyper-Raman spectroscopy typically is performed using SERS-active or NERS-active structures.
One limitation inherent in heretofore known Raman spectroscopy systems is the inability to focus the incident electromagnetic radiation to a “spot size” having linear measurements less than about one wavelength of the electromagnetic radiation due to the diffraction limit. As Raman spectroscopy typically is performed using incident electromagnetic radiation in or near the visible region of the electromagnetic spectrum, the incident electromagnetic radiation generally cannot be focused to a spot size smaller than about 100 nanometers in diameter. This inability to provide a relatively smaller spot size precludes performing Raman spectroscopy on smaller, more precise regions of an analyte. It is desirable to provide ever smaller spot sizes, which theoretically could be used to conduct Raman spectroscopy on a particular portion of a molecule, such as, individual nucleotides of a DNA molecule or particular amino acids or peptides of a protein. Therefore, it is desirable to provide Raman spectroscopy systems configured to emit incident electromagnetic radiation to ever smaller spot sizes.